Glycoproteins are involved in a wide variety of multivalent interactions that are of physiological importance. The many potential roles of carbohydrates covalently attached to proteins are of interest in theoretical structure-function studies and practical applications. In particular, applications such as increasing the stability and solubility of proteins (Sundaram, P. V. and Venkatesh, R., 1998, Protein Eng., 11, 699-705, Ed.), (Aoki, T., Hiidome, Y., Kitahata, K., Sugimoto, Y., Ibrahim, H. R. and Kato, Y., 1999, Food Res. Int., 32, 129-133, Ed.) and the development of vaccines (Mammen, M., Choi, S.-K. and Whitesides, G., 1998, Angew. Chem. Int. Ed., 37, 2754-2794, Ed.), (Wong, S. Y., 1995, Current Opin. Struct. Biol., 5, 559-604, Ed.), (Roy, R., 1996, Current Opin. Struct. Biol., 6, 692-702, Ed.) have spurred efforts to discover efficient methods of chemical attachment of carbohydrate to proteins, i.e. glycation. Protein glycation is often carried out under aqueous conditions where the reaction is unfavorable due to the fact that water is a product. Current aqueous glycation methods also require relatively large amounts of protein and carbohydrate (Yeboah, F. K., Alli, I. and Yaylayan, V. A., 1999, J. Agric. Food Chem., 47, 3164-3172, Ed.), (Roy, R., Katzenellenbogen, E. and Jennings, H. J., 1984, Can. J. Biochem. Cell Biol., 62, 270-275, Ed.), and the extent of glycation is very difficult to control (Wrodnigg, T. M. and Eder, B., 2001, Glycoscience, 215, 115-152, Stutz, A. E., Ed., Springer-Verlag). Another significant drawback is that the desired glycation product, cyclic ketoamine, is contaminated with advanced glycation products (Wrodnigg, T. M. and Eder, B., 2001, Glycoscience, 215, 115-152, Stutz, A. E., Ed., Springer-Verlag), (Yaylayan, V. A. and Huyghues-Despointes, A., 1994, Crit. Rev. Food Sci. Nutrition, 34, 321-369, Ed.) i.e Maillard browning reaction.
An effective method of increasing the efficiency of a reaction is by the removal of a product. The first step in the glycation of an amino group is believed to be the reaction of a deprotonated amine with the aldehyde group of a reducing sugar yielding water and a Schiff base as products (Yaylayan, V. A. and Huyghues-Despointes, A., 1994, Crit. Rev. Food Sci. Nutrition, 34, 321-369, Ed.), (Wrodnigg, T. M. and Eder, B., 2001, Glycoscience, 215, 115-152, Stutz, A. E., Ed., Springer-Verlag). In aqueous media, formation of the Schiff base is reversible, and in situ reductive alkylation of the Schiff base forming a stable derivative has been employed to achieve efficient glycation (Cayot, P., Roullier, L. and Tainturier, G., 1999, J. of Agric. & Food Chem., 47, 1915-1923, Ed.), (Wrodnigg, T. M. and Eder, B., 2001, Glycoscience, 215, 115-152, Stutz, A. E., Ed., Springer-Verlag). Theoretically, another way of promoting the glycation reaction is by the removal of the water but there is no obvious experimental approach for such a strategy under aqueous conditions. Water could be removed by carrying out the reaction in the dry state under vacuum. Dry state glycation has been attempted under a variety of conditions (Boratynski, J. and Roy, R., 1998, Glycoconjugate J., 15, 131-138, Ed.), (Boratynski, J., 1998, Biotechnol. Tech., 12, 707-710, Ed.), (Quan, C. P., Wu, S., Dasovich, N., Hsu, C., Patapoff, T. and Canova-Davis, E., 1999, Anal. Chem., 71, 4445-4454, Ed.), (Morgan, F., Leonil, J., Molle, D. and Bouhallab, S., 1999, J. Agric. Food Chem., 47, 83-91, Ed.), (Yeboah, F. K., Alli, I., 1999, J. Agric. Food Chem., 47, 3164-3172, Ed.), (French, S. J., Harper, W. J., Kleinholz, N. M., Jones, R. B. and Green-Church, K. B., 2002, J. Agric. Food Chem., 50, 820-823, Ed.) but a common observation is that many glycation products are observed and the mechanism of their formation is unclear. None of these studies have used a vacuum to promote the glycation reaction by the removal of water or to prevent the formation of advanced glycation end products.
Amino groups in dry proteins are present in their protonated form, and for glycation to take place, the reaction would have to involve these protonated amino groups. On the basis of current theory, a protonated amino group in solution does not react with the aldehyde form of a reducing sugar. Furthermore, there is no known theory that predicts that if a mixture of a protein and reducing sugar in the dry state were subjected to a vacuum that a water-stable glycated derivative would be formed. The fact that extensive glycation of proteins does occur in the lyophilized state under vacuum with heating demonstrates that the protonated amino group does indeed react. Therefore, there are two novel theoretical features to the discovery that lyophilized proteins can be efficiently glycated in vacuo in the dry state: 1. A protonated amino group will react with a sugar aldehyde group in vacuo. 2. A ketoamine derivative is formed which does not rapidly revert to the free amine and sugar when placed in aqueous solution.
Covalent cross-linking of proteins is a major tool for the study of structure-function relationships in proteins and has many practical applications (Fancy, D. A., 2001, Curr. Opin. Chem. Biol., 4, 28-33, Ed.), (Phizicky, E. M., 1995, Microbiol. Rev., 59, 94-123, Ed.), (Lundblad, R., 1994, Techniques in Protein Modification, 249-261, Ed., CRC Press). Homo-bifunctional reagents with variable spacing between the reactive groups have been widely used to achieve such cross-links (Lundblad, R., 1994, Techniques in Protein Modification, 249-261, Ed., CRC Press). To our knowledge, reducing sugars have never been used as bifunctional reagents for the cross-linking of proteins. However, the discovery of the facile glycation that occurs in vacuo indicates that a reagent with two or more reducing sugars with variable spacing could be used to covalently cross-link proteins in the lyophilized state. The ideal number of spacer units separating the reducing sugars depends on the protein or proteins being cross-linked. In general, the ideal number of units will likely be less than 10 but in some applications could be much greater. The cross-linking methodology would be identical to the in vacuo glycation with reducing sugars.
In vacuo glycation is easier to carry out than aqueous glycation. It requires only co-lyophilization of appropriate amounts of protein and reducing sugar followed by incubation at an elevated temperature under vacuum. In addition, in vacuo glycation has several other significant technical advantages.    1. The procedure can be carried out using a wide range of protein and/or carbohydrate quantities, viz. gram to picogram quantities.    2. The protein can be lyophilized at a pH value where it retains its native structure and biological activity.    3. The extent of glycation is easily controlled either by adjusting the protein/carbohydrate ratio or by the addition of excipients.    4. Elevated temperatures can be used to increase the rate of glycation without structural damage to the protein or carbohydrate.    5. With proteins, no contaminating advanced glycation products (Maillard browning reaction) are observed. Only the ketoamine derivative is observed.    6. Complex carbohydrates or compounds containing two or more reducing sugars can be used to cross-link proteins.
There is therefore a need for a facile method of glycating proteins.